[0001] The present invention relates to a treatment of cancer combining enzymatic with metabolic
means of selective attack on cancer cells.
Background
[0003] The use of arginine-depleting enzymes is necessary, but our own research has shown
it is not sufficient to cause and maintain systemic, deep depletion of arginine needed
to cause rapid, selective killing of cancer cells.
[0004] The use of an insulin/glucose clamp in parallel with the enzymatic degradation of
arginine makes the task of deep arginine depletion much more manageable. Insulin is
a growth factor and thus promotes protein synthesis and inhibits protein breakdown.
This is of crucial importance when the task is removing an amino acid from circulation,
particularly removing arginine, which is a semi-essential amino acid under tight homeostatic
control.
[0005] An increase of vascular permeability by insulin also helps in getting therapeutic
enzymes into interstitial fluid space, closer to where most cancerous cells reside.
Finally, insulin may also play a role in transporting arginine-degrading enzymes into
cancerous cells by stimulating endocytosis.
[0006] However, as the inventors' research has shown, even with the use of an insulin/glucose
clamp, free arginine levels could only be reduced to about 5 to 10 µM, i.e., to about
5 to 10% of the normal plasma concentration of about 100 µM. At these free arginine
levels, cells, cancerous and healthy, will not proliferate but still can survive for
prolonged periods. The target for arginine concentration to result in rapid killing
of cancer cells is 1 µM or less. Healthy cells can survive such low levels of arginine
much longer than cancer cells (weeks vs days) which accounts for the selectivity of
this intervention.
[0007] The main obstacle to achieving this level of arginine depletion is the conversion
of citrulline into arginine by the kidneys. Citrulline is produced by intestinal lining
cells from ornithine which in turn is produced from non-essential amino acids glutamine
and proline.
[0008] The inventors have attempted inhibition of citrulline production in the intestines
but none of the many approaches tested in experimental dogs have resulted in the satisfactory
reduction of plasma citrulline.
[0009] The resolution to the problem of citrulline as a precursor of arginine was found
in the use of argininosuccinate synthase (ASS) which converts citrulline and aspartate
to argininosuccinate. ASS is delivered to the vascular system where it interrupts
the transport of citrulline from the intestines to the kidneys. In the urea cycle
of the liver, argininosuccinate is converted to arginine by argininosuccinate lyase
(ASL) but this does not happen in the vascular system. Accordingly,
WO 2023/066910, the content of which is herein incorporated by reference, describes a medicament
comprising an arginine decomposing enzyme such as arginase (ARG) and a citrulline
converting enzyme such as argininosuccinate synthase (ASS) for the treatment of cancer.
[0010] Certain blood cancers, e.g., acute lymphoblastic leukemia (ALL) or non-Hodgkin lymphoma,
are highly susceptible to depletion of asparagine, a non-essential amino acid. The
use of L-asparaginase (ASNase) in treating these blood cancers is a highly successful
precedent for all other amino-acid depletion schemes.
[0011] Unlike arginine, asparagine is easily depleted from circulation for the apparent
lack of any systemic mechanisms of homeostasis. L-asparaginase also degrades glutamine,
another non-essential amino acid. Glutamine is used as a precursor for many biosynthetic
pathways and is found in abundance in all cells and in all extracellular fluids.
[0012] WO 2020/245041, the content of which is herein incorporated by reference, discloses delivery of
asparaginase (ASNase) in a dissociated form, particularly in the form of monomeric
units.
[0013] WO 2022/034218, the content of which is herein incorporated by reference, discloses that the efficacy
of biologics may be increased by assisting the extravasation of these compounds by
co-administering a biological therapeutic molecule together with insulin and glucose.
[0014] It was an object of the present invention to provide means for improving the depletion
of free arginine in the blood of a subject, particularly of a cancer patient. More
particularly, it was an object of that invention to overcome disadvantages associated
with previous treatment schedules involving amino acid depletion with, e.g., administration
of PEGylated arginase (ARG) or PEGylated arginine deiminase (ADI), which are currently
investigated in over 30 clinical trials for cancer treatment.
Summary of the invention
[0015] The present inventors have found that the combined catalytic activity of these three
enzymes ARG, ASS, and ASNase together with insulin but under conditions of glucose
depletion leads to the systemic reduction of arginine, citrulline, asparagine, and
glutamine. Most cancer cells are rapidly - in a matter of one or a few days - eliminated
while the healthy cells survive with minimal losses. Healthy, normally proliferating
cells such as those of the intestinal lining or bone marrow, exit the cycle but can
resume proliferation once these amino acids are brought back to normal physiological
levels.
[0016] A first aspect of the present invention relates to an amino acid-degrading enzyme
for use in medicine, wherein a subject in need thereof is subjected to a treatment
with at least one amino acid-degrading enzyme and a blood glucose-lowering agent under
conditions of glucose depletion.
[0017] A further aspect of the present invention relates to a method for the treatment of
cancer wherein a subject in need thereof is subjected to a treatment with at least
one amino acid-degrading enzyme and a blood glucose-lowering agent under conditions
of glucose depletion.
[0018] In certain embodiments, the amino acid-degrading enzyme is selected from:
- an asparaginase, particularly in a dissociated form, particularly in the form of monomers;
- an arginase;
- an argininosuccinate synthase;
- a glutaminase;
- or any combination thereof.
[0019] The present invention combines two powerful attacks on cancer cells - systemic depletion
of amino acids for protein synthesis and depletion of glucose. In particular, the
amino acids targeted are arginine and glutamine, together with citrulline and asparagine.
The preferred enzyme for the degradation of arginine is arginase (ARG). The preferred
enzyme for the degradation of glutamine is asparaginase (ASNase), which converts asparagine
to aspartic acid but also glutamine to glutamic acid.
[0020] Our experimental clinical studies with ASNase for canine lymphoma have shown variable
effectiveness of ASNase in lowering systemic levels of glutamine. It is therefore
an option to use glutaminase as the fourth enzyme. Our recent in vitro research gives
a strong rationale for combining these enzymes. Exposed to arginase, cancer cells
have shown decreased intracellular concentrations of all amino acids but of asparagine
and glutamine that have increased. While it is not clear why, it seems reasonable
to assume that removing these two amino acids with asparaginase and glutaminase should
have a synergetic effect with arginase.
[0021] Further, glucose is systemically lowered by the use of a glucose-lowering agent such
as insulin.
[0022] Furthermore, the lack of glucose may be supplemented by administration of a ketone
body compound.
Description of preferred embodiments
[0023] According to the present invention, a subject in need thereof is subjected to a treatment
with at least one amino acid-degrading enzyme and a blood glucose-lowering agent under
conditions of glucose depletion. The term "amino acid-degrading enzyme" relates to
an enzyme which is capable of reducing the amount of an amino acid under physiological
conditions. In certain embodiments, the present invention comprises a treatment involving
reducing the amount of the amino acids arginine, citrulline, asparagine and glutamine
in order to provide conditions which are detrimental for the survival of cancer cells
within the body of a subject to be treated.
[0024] In certain embodiments, the present invention relates to the depletion of arginine
by enzymatic means for use in medicine, particularly for the treatment of cancer,
in combination with the concurrent depletion of glucose and glutamine.
[0025] The inventors have found that systemic delivery of an arginine-decomposing enzyme
such as an arginase (ARG) and a citrulline-converting enzyme such as argininosuccinate
synthase (ASS) in partially purified liver extracts prepared by low-temperature protocols
has led to a reduction of free plasma arginine to below detection levels. This was
the basis of co-owned application
WO 2023/066910, supra, describing a medicament comprising an arginine-decomposing enzyme such as
an ARG and a citrulline-converting enzyme such as an ASS for use in a deep depletion
of arginine levels in circulating blood.
[0026] Enzymatic decomposition of arginine by e.g., an arginase (ARG) was found to be necessary
but not sufficient for a deep, systemic depletion of arginine. A concurrent removal
of citrulline on its passage from intestines, the main organ for citrulline synthesis,
to kidneys where citrulline is converted to arginine, by a citrulline-converting enzyme,
e.g., argininosuccinate synthase (ASS) enables systemic reduction of free arginine
to a concentration below detection by standard amino acids analysis. This leads to
the rapid killing of cancerous cells of many if not all cancer types. Healthy dividing
cells respond by exiting into G
0 phase and very few suffer terminal damage.
[0027] In certain embodiments, the arginine-decomposing enzyme is an arginase (ARG), i.e.,
an enzyme, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea (EC.
3.5.3.1). Typically, the arginase is a mammalian arginase, e.g., a human arginase
including any enzymatically active fragment and derivative thereof. In particular
embodiments, the arginase is selected from human arginase-1 (liver arginase or ARG1)
(UniProt-P05089) or human arginase 2 (kidney arginase or ARG2) (UniProt-P78540) including
any enzymatically active fragment and derivative thereof. In even more particular
embodiments, the arginase is human ARG1.
[0029] In certain embodiments, the arginase is a recombinant human ARG1 including any enzymatically
active fragment and derivative thereof. In particular embodiments, the arginase is
an unPEGylated polypeptide, i.e., it is not conjugated to a polyethylene glycol moiety.
[0031] In certain embodiments, arginase, e.g., human ARG1 is administered as an arginase-insulin
fusion protein as described in co-owned application
WO 2023/041758, the content of which is herein incorporated by reference.
[0032] In another particular embodiment, the arginine-decomposing enzyme is an arginine
deiminase, e.g., an arginine deiminase of 46 kDa (UniProt-A0A0C6G6L6_MYCAR).
[0033] In another particular embodiment, the arginine-decomposing enzyme is an arginine
decarboxylase, e.g., a biosynthetic arginine decarboxylase of 74 kDa (UniProt-Q8FE34
SPEA_ECOL6) or a biodegradative arginine decarboxylase of 80 kDa (UniProt-A0A376VTJ3_ECOLX).
[0034] Arginase may be supplied externally, but an alternative is its endogenous release
from the liver which would, due to low glucose, undergo a degree of necrosis and/or
apoptosis.
[0035] In certain embodiments, the citrulline-converting enzyme is an argininosuccinate
synthase (ASS), i.e., an enzyme, which catalyzes the conversion of L-aspartate and
L-citrulline to L-argininosuccinate (EC 6.3.4.5). Typically, the ASS is a mammalian
ASS, e.g., a human ASS including any enzymatically active fragment and derivative
thereof. In particular embodiments, the ASS is human argininosuccinate synthase (ASS1)
(UniProt-P00966) including any enzymatically active fragment and derivative thereof.
[0036] Human ASS1 is a 412 amino acids long polypeptide with a predicted molecular weight
of 46,530 Da. It may be present in a monomeric or multimeric, e.g., tetrameric form.
Preferably, it is present in a tetrameric form, which is its native active form in
the human liver.
[0037] In certain embodiments, the ASS is a recombinant human ASS1 including any enzymatically
active fragment and derivative thereof. In certain embodiments, the ASS is a PEGylated
polypeptide. In other embodiments, the ASS is an unPEGylated polypeptide.
[0038] In certain embodiments, ASS is needed to bring arginine to a very low concentration.
It also is released by the liver due to the gradual, partial loss of hepatocytes deprived
of glucose. ASS may be supplied externally, but an alternative is its endogenous release
from the liver.
[0039] In certain embodiments, ARG and ASS are present as at least partially purified mammalian
liver extract. A preferred method for obtaining a suitable liver extract is described
in the Examples of the co-owned application
WO 2023/066910, supra. In certain embodiments, ARG and ASS are recombinant polypeptides.
[0040] In certain embodiments, both ARG and ASS are supplied by external administration,
e.g., infusion.
[0041] In certain embodiments, an external administration of ARG and/or ASS is not required.
[0042] Another aspect of research by the inventors was the modification of the delivery
of L-asparaginase (ASNase). ASNase can be purified from certain strains of
E.
coli or can alternatively be produced by recombinant technology from genetically modified
strains of
E.
coli. ASNase is a commonly used drug to treat blood cancers, e.g., leukemias and lymphomas.
[0043] In a particular embodiment, the ASNase its in its tetrameric (active) form of 140
kDa, or in its monomeric form of 35 kDa.
[0044] In an even more particular embodiment, the ASNase is in a dissociated form, particularly
in the form of a monomer. The reversible dissociation of ASNase into its monomers
by a high concentration of a chaotropic agent such as urea as a functional excipient
facilitates the extravasation of monomers which are then reconstituted in the interstitial
fluid in the surrounding of most of the cancer cells of any type of cancer, including
blood cancers, as disclosed in the co-owned application
WO 2020/245051, supra.
[0045] The asparaginase may be administered as an aqueous preparation comprising urea, particularly
in a concentration between about 3 mol/l to about 8 mol/l, more particularly in a
concentration of about 4 mol/l to about 6 mol/l, e.g., about 5 mol/l.
[0046] Further, the present invention comprises administration of a blood glucose-lowering
agent, e.g., an insulin or an insulinotropic peptide, particularly a fast-acting insulin.
Insulin is a growth factor that also is a glucose-lowering hormone.
[0047] In certain embodiments, the insulin is administered as an arginase-insulin fusion
protein as described as described in co-owned application
WO 2023/041758, supra.
[0048] The treatment according to the present invention is carried out under conditions
of glucose depletion. Glucose depletion is understood as a blood glucose level of
lower than the normal glucose level of the subject to be treated. In certain embodiments,
conditions of glucose depletion involve adjusting and maintaining a blood glucose
level of about 2 mM or less, e.g., about 0.8 mM to about preferably to as low as about
1 mM. Glucose depletion may be achieved by administration of a blood glucose-lowering
agent as described herein.
[0049] In certain embodiments, conditions of glucose depletion are maintained throughout
a treatment cycle, e.g., during the administration of the amino acid-degrading enzyme.
In certain embodiments, the conditions of glucose depletion are maintained for at
least 12 h, at least 24 or at least 48 h.
[0050] In certain embodiments, the blood glucose-lowering agent is administered without
concomitant administration of glucose or a glucose precursor, e.g., a glucose-containing
oligo or polysaccharide.
[0051] Furthermore, the present invention may comprise the administration of a ketone body
compound.
[0052] The brain can use ketone bodies and its glial cells are also capable of producing
ketone. During the treatment, most of the energy needs of the healthy cells are fulfilled
by the administration, e.g., infusion of ketone bodies, preferably in the form of
3-hydroxybutyric acid, or a physiologically acceptable 3-hydroxybutyrate salt, e.g.,
an alkaline metal, alkaline earth metal or amino acid salt. In certain embodiments,
the salt is selected from sodium, potassium, calcium, and magnesium 3-hydroxybutyrate
or a combination thereof. The use of a balanced mixture of those salts may avoid the
overloading of sodium while providing the needed amount of butyrate. Administering
an amino acid salt, e.g., lysine 3-hydroxybutyrate, is of special interest since lysine
is an antagonist of arginine. An alternative to infusion is exogenous oral delivery.
Residual systemic glucose usually provides the minimum needed to support the brain,
liver, and erythrocytes.
[0053] In certain embodiments, glutaminase (GLS) is administered, particularly in combination
with ASNase and optionally ARG and/or ASS. GLS can be added should ASNase fail to
sufficiently lower glutamine. In dogs treated with asparaginase, glutamine concentration
could be lowered to between the detection limit (<1 micromolar) and 100 micromolar,
from the normal level of close to 1 mM. An effective reduction calls for glutamine
of < 50 micromolar, preferably of < 10 micromolar.
[0054] In certain embodiments, the GLS is a kidney-type GLS, e.g., human kidney (GLSK) with
669 amino acids and a molecular weight of 73,461 Da or any processed form thereof
such as Isoform 3 of 68 kDa, or a liver-type GLS, e.g., human liver GLS (LGA)as described
by
Katt et al (Future Med Chem 2017, 9(2), 223-243, and 9(5), 527), the content of which is herein incorporated by reference In certain
embodiments, the GLS is a recombinant polypeptide.
[0055] A particular aspect of the invention relates to a medicament comprising the following
active agents:
- (i) an asparagine-decomposing enzyme such as an ASNase;.
- (ii) a blood glucose-lowering agent, e.g., an insulin;
- (iii) a ketone body compound, e.g., a physiologically acceptable 3-hydroxybutyrate
salt;
- (iv) an arginine-decomposing enzyme such as an ARG;
- (v) a citrulline-converting enzyme such as an ASS, and
- (vi) optionally a glutamine-decomposing enzyme such as a GLS.
[0056] In certain embodiments, ARG and/or ASS need not be added externally but are generated
endogenously as described above.
[0057] The medicament of the invention is suitable for use in human medicine and veterinary
medicine, e.g., for the treatment of dogs. While the use of the human versions of
ARG and ASS is acceptable in treating dogs, canine versions of ARG and ASS are preferred.
Both have been produced as recombinant proteins expressly for use in dogs by the present
inventors.
[0058] Administration of the medicament in a therapeutically active dose will lead to a
deep depletion of arginine levels in the blood. In certain embodiments, the arginine
levels are about 20 µM or less for a time of at least 12 h or even for at least 72
h, preferably the arginine levels are about 10 µM or less for a time of at least 12
h or even for at least 72 h, more preferably the arginine levels are about 5 µM or
less for a time of at least 12 h or even for at least 72 h, and most preferably the
arginine levels are about 1 µM or less for a time of at least 12 h or even for at
least 72 h as measured in the venous blood plasma. Citrulline concentration in the
human plasma of healthy individuals is 30 to 50 µM. Observations from human patients
with advanced hepatocellular carcinoma that the inventors participated in treating
with arterial occlusion of the liver suggest that lowering citrulline level two to
five-fold is sufficient to lower arginine level to the target of less than 1 µM. In
dogs normal citrulline concentration is about two times higher than in humans but
the same reduction of two to five times was correlated with successful arginine depletion.
[0059] In certain embodiments, the medicament of the present invention comprises five active
agents. Three are amino acid degrading enzymes, one or two of which (ARG and ASS)
may be added externally or released endogenously from the liver deprived of the minimum
required circulating glucose. L-asparaginase (ASNase) is the first active agent and
in all cases it is supplied externally by infusion. The second active agent is a blood
glucose-lowering agent such as insulin. The third is a ketone body compound, preferably
3-hydroxybutyrate or a physiologically acceptable salt thereof such as sodium 3-hydroxybutyrate.
The fourth and the fifth active agents are ARG and ASS, respectively. The active agents
are administered together with pharmaceutically acceptable excipients, e.g., selected
from buffers, salts, and/or stabilizers
[0060] The medicament may be a pharmaceutical preparation comprising each of the active
agents separately. Alternatively, the medicament may be a combination of three separate
pharmaceutical preparations, one comprising ASNase dissolved in urea, the other comprising
ARG and ASS, and the third comprising insulin added to a solution of a ketone body
compound such as 3-hydroxybutyrate.
[0061] The pharmaceutical preparation may be a liquid pharmaceutical preparation comprising
the active agent(s) in dissolved or suspended form ready for use.
[0062] Alternatively, the pharmaceutical preparation may be a solid pharmaceutical preparation
comprising the active agent(s) in lyophilized or freeze-dried form for reconstitution
with a suitable solvent, e.g., an aqueous solvent.
[0063] The medicament is administered in therapeutically effective doses of the active agents.
[0064] Asparaginase (ASNase) in conventional clinical use for blood cancers in people and
in dogs is typically given at a dose of 400 IU/kg, 3 times a week for about 3 months.
Our own research has demonstrated that this is greatly inadequate. To reach the levels
of the enzyme in the interstitial fluid (ISF) needed to exert a relevant effect on
the cancer cells based on what is known from the dose-response studies
in vitro we have been using in experimental and clinical studies in animals 3000 IU/kg. To
maintain the enzymatic activity in the ISF, a daily dose of at least about 1000 IU/kg,
e.g., about 3000 IU/kg, may be delivered to the subject in need thereof, e.g., a huma
or dog. The daily dose is preferably delivered by a continuous infusion, e.g., for
the duration of the treatment of 2 to 4 days and no longer than 6 days depending on
the type of cancer.
[0065] In certain embodiments of the invention, the systemic concentration of glucose is
lowered by infusion of insulin. The goal is to lower plasma glucose concentration
to the minimum level needed to supplement ketone bodies delivered by infusion. In
healthy individuals on a regular diet, the glucose level is about 5 mM while the ketone
level is about 0.5 to 1 mM. The ratio of glucose to ketone (so-called glucose-ketone
index, or GKI) is thus about 5 to 10. Infusion of ketone can safely bring its plasma
concentration to 4 or 5 mM. Insulin should be used to lower glucose to below about
2 mM, preferably to as low as about 1 mM, inverting the ratio of the two basic sources
of energy. Thus, in certain embodiments, GKI in blood is about 1 or less, e.g., about
0.1 to about 0.5.
[0066] The insulin may be administered by infusion at a predetermined dose rate, e.g., at
a predetermined constant dose rate. For humans, the insulin may be administered at
a dose rate from about 0.2 to about 2 IU/kg body weight/day, preferably from about
0.5 to about 1.5 IU/kg body weight//day, more preferably about 1 IU/kg body weight//day.
For dogs, the insulin may be administered at a higher dose rate from about 0.4 to
about 4 IU/kg body weight//day, preferably from about 1 to about 3 IU/kg body weight//day,
more preferably about 2 IU/kg body weight//day.
[0067] The insulin may be any type of natural or recombinant insulin or insulin analogue.
Preferably, the insulin is a rapid acting insulin or a short acting insulin, more
preferably a rapid acting insulin. Examples of rapid acting insulin are insulin lispro,
insulin aspart or insulin glulisine. Examples of short acting insulins are regular
insulin or insulin velosulin.
[0068] Ketone bodies, particularly sodium 3-hydroxybutyrate, have been infused for different
pathological conditions but also experimentally in studies of heart performance. Infused
at a rate of about 0.2 g/kg/hour, its plasma concentration was increased from zero
to between 3 and 4 mM.
[0069] In certain embodiments, the therapeutically effective daily dose of an arginase,
e.g., ARG1, in a dog is from about 300 to about 6000 IU/kg/day, preferably from about
1000 to about 4500 IU/kg/day, and most preferably 3000 IU/kg/day as determined by
experimental work
in vivo. For humans, a therapeutically effective daily dose is about 150 to about 3000 IU/kg/day,
preferably about 500 to about 2000 IU/kg/day, and most preferably about 1500 IU/kg/day.
With a specific activity of recombinant ARG1 of about 1000 IU/mg of protein a preferred
therapeutically effective daily dose in a dog is about 0.3 to about 6.0 mg/kg/day
and in a human about 0.15 to about 3.0 mg/kg/day.
[0070] In certain embodiments, the therapeutically effective daily dose of an ASS, e.g.,
ASS1, in a dog is from about 0.3 to about 6 IU/kg/day, preferably from about 1 to
about 4.5 IU/kg/day, and most preferably 3 IU/kg/day. For humans, a therapeutically
effective daily dose is about 0.15 to about 3 IU/kg/day, preferably about 0.5 to about
2 IU/kg/day, and most preferably about 1.5 IU/kg/day. With a specific activity of
recombinant ASS1 of about 1 IU/mg of protein a preferred therapeutically effective
daily dose in a dog is about 0.3 to about 6.0 mg/kg/day and in a human about 0.15
to about 3.0 mg/kg/day.
[0071] In certain embodiments, the dose is adapted based on the measurement of arginine
and/or citrulline levels in the blood of the subject to be treated. In particular
embodiments, the arginine and/or citrulline levels are measured in the plasma of the
venous blood.
[0072] The medicament is typically administered parenterally, e.g., by injection, or preferably
by infusion over a suitable period, for example, over a period from several hours,
e.g., at least about 2 hours, or about 6 hours, or even continuously during 1 day
up to 6 days, depending on the type of cancer being treated.
[0073] Administration of the medicament may be accompanied by certain measures to compensate
for side-effects of arginine depletion such as infusion of a nitric oxide (NO) donor,
e.g., sodium nitroprusside (SNP), and/or a pressor peptide, e.g., a vasopressin, to
balance NO-induced vasodilation. Arginine is the only precursor for the synthesis
of short-lived NO. All pressor peptides contain arginine and are short-lived. Co-infusion
of Iloprost, a prostacyclin analog has also been found useful in the maintenance of
thrombocytes. Infusion of glucose is used only in an emergency should any clinical
signs of its lack become manifest.
[0074] The medicament is useful for the treatment of cancer including a blood cancer such
as leukemia and lymphoma, or a solid cancer particularly selected from liver cancer
including primary liver cancer and hepatocellular carcinoma, skin cancer such as melanoma,
colon carcinoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, pancreatic cancer,
lung cancer, ovarian cancer, prostate cancer, gastric cancer and breast cancer. The
medicament is also useful for the treatment of cancer metastases, which are disseminated
within the patient's body.
[0075] The inventors have found that the efficacy of the medicament may be increased by
assisting extravasation of arginase, i.e., the transport from the vascular system
into the interstitial fluid. Rapid extravasation of ARG1 in its monomeric form, facilitated
by insulin, prevents its elimination by glomerular filtration as described in co-owned
application
WO 2022/034218 the content of which is herein incorporated by reference.
[0076] In certain embodiments, the amino acid-degrading enzyme may be administered to the
subject under conditions which promote protein transport into the interstitial fluid.
In particular embodiments, the treatment comprises administration of at least amino
acid-degrading enzyme under conditions of blood and/or plasma volume expansion. Such
a treatment is disclosed in co-owned application
EP 23 200 562.9, the content of which is herein incorporated by reference.
[0077] The target compartment for the ARG and ASNase is the interstitial volume. This is
where most cancer cells reside - only a small fraction of cancer cells are found in
the blood at any stage of the disease, even in blood cancers. Enzymatic activity confined
to the vascular system cannot efficiently reduce concentrations of amino acids in
extravascular volume to very low levels because of the constant influx of amino acids
from protein breakdown under homoeostatic mechanisms. The mass transport between intravascular
and interstitial fluid (in both directions) is limited by diffusion and modest convection,
the rates of which are simply too low when, e.g., arginine is to be systemically reduced
into µM range. By contrast, ASS is preferably retained in the blood to intercept the
transport of citrulline from the intestines to the kidneys. Its large molecular weight
of 186 kDa helps to keep it in the vascular system. Optionally, it could be PEGylated.
[0078] It is worth noting that all current clinical trials with arginine depletion as a
modality for cancer treatment are conducted with PEGylated enzymes (arginase or arginine
deiminase) with high molecular weights that make extravasation extremely limited.
This prevents deep systemic arginine depletion needed to kill disseminated cancer.
There is also a rapid transition to PEGylated ASNase in some of the markets. This
is a very unfortunate, misguided development that makes the problem of ASNase underdosing
even worse.
[0079] According to the present invention, insulin may be co-administered with ketone bodies,
preferably with sodium 3-hydroxybutyrate. Preferably, hydroxybutyrate is administered
together with insulin by continuous infusion throughout the treatment period, optionally
by oral intake.
1. An amino acid-degrading enzyme for use in medicine,
wherein a subject in need thereof is subjected to a treatment with at least one amino
acid-degrading enzyme and a blood glucose-lowering agent under conditions of glucose
depletion.
2. The amino acid-degrading enzyme for the use of claim 1, wherein the at least one amino
acid-degrading enzyme comprises an asparaginase, particularly an asparaginase in a
dissociated form, more particularly in the form of monomers.
3. The amino acid-degrading enzyme for the use of claim 2, wherein the at least one amino
acid-degrading enzyme comprises an asparaginase preparation comprising a pharmaceutically
acceptable chaotropic agent is administered, wherein the chaotropic agent is particularly
selected from urea, more particularly urea in a concentration of about 3 mol/l to
about 8 mol/l, even more particularly in a concentration of about 4 mol/l to about
6 mol/l, e.g., about 5 mol/l.
4. The amino acid-degrading enzyme for the use of any one of claims 1-3, wherein the
treatment with at least one amino acid-degrading enzyme comprises a treatment with
(i) an asparaginase;
(ii) an arginase;
(iii) an argininosuccinate synthase; and
(iv) optionally a glutaminase,
wherein the arginase (ii) and/or the argininosuccinate synthase (iii) are administered
externally or provided endogenously.
5. The amino acid-degrading enzyme for the use of claim 4, wherein the asparaginase,
the arginase, and the argininosuccinate synthase are administered externally.
6. The amino acid-degrading enzyme for the use of any one of claims 1-5, wherein the
blood glucose-lowering agent is an insulin or an insulinotropic peptide, particularly
a fast-acting insulin.
7. The amino acid-degrading enzyme for the use of any one of claims 1-6, wherein the
treatment further comprises administration of a ketone body compound, e.g., a physiologically
acceptable 3-hydroxybutyrate salt.
8. The amino acid-degrading enzyme for the use of any one of claims 1-7, wherein the
treatment comprises administration of at least amino acid-degrading enzyme by intravenous
infusion.
9. The amino acid-degrading enzyme for the use of any one of claims 1-8, wherein the
treatment comprises administration of at least one amino acid-degrading enzyme is
administered to the subject under conditions which promote protein transport into
the interstitial fluid.
10. The amino acid-degrading enzyme for the use of any one of claims 1-9, wherein the
treatment comprises administration of at least amino acid-degrading enzyme under conditions
of blood and/or plasma volume expansion.
11. The amino acid-degrading enzyme for the use of any one of claims 1-10, for use in
the treatment of cancer.
12. The amino acid-degrading enzyme for the use of claim 11, wherein the cancer is selected
from a blood cancer such as leukemia and lymphoma, or a solid cancer such as liver
cancer including primary liver cancer, skin cancer such as melanoma, breast cancer,
colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and
gastric cancer.
13. The amino acid-degrading enzyme for the use of any one of claims 1-12, wherein the
subject is a human.
14. The amino acid-degrading enzyme for the use of any one of claims 1-12, wherein the
subject is a non-human mammal, particularly a cat or dog.
15. A method for the treatment of cancer wherein a subject in need thereof is subjected
to a treatment with at least one amino acid-degrading enzyme and a blood glucose-lowering
agent under conditions of glucose depletion.